Serotonin binding protein (SBP) is a protein that specifically binds serotonin with high affinity under intracellular ionic conditions but which releases bound serotonin when exposed to estracellular concentration of Na+ and Ca+2. The protein has been associated with neurectodermally-derived cells that store serotonin and considerable evidence suggests that it is a component of synaptic vesicles . Two forms of SBP (45K and 56K daltons) have been identified. We have proposed the hypothesis that SBP and serotonin are stored together as a macromolecular complex in synaptic vesicles. We now wish to confirm anatomically whether SBP and serotonin are in fact stored in the same cells and vesicles. This will be done by raising monospecific antibodies to 45K and/or 56K SBP. These antibodies, and antibodies to serotonin, will be used to locate simultaneously the protein and the amine in the same tissue sections by immunocyto-chemistry. Immunocytochemistry will also be used at the electron microscopic level to explore the subcellular localization of SBP. In additional experiments the relationship distribution and characteristics of the 45K and 56K forms of SBP will be analyzed; the possible derivation from SBP of peptides, such as substance P and TRH, that are co-stored with serotonin by proteolytic nicking of SBP will be investigated; the effect of dietary conditions (Fe+2-deficient; low tryptophan, high tryptophan) that effect binding of serotonin by SBP on in vivo storage of the amine will be studied; finally, the ontogeny of SBP will be examined in correlation with the development of synaptic vesicles and the ability of neurons to take up and store serotonin. These experiments are designed to provide insight into molecular mechanisms involved in serotonin storage. Such insight would be expected to be most helpful in understanding conditions, such as the affective disorders, that may arise because of abnormal serotonergic function and in the development of new means of therapeutic intervention.